Etiologic Risk Group (see link below):Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents
Disease Information: Toxoplasmosis is a resourceful and global infection that results from the intracellular protozoan parasite Toxoplasma gondii. The disease was discovered by scientists researching in North Africa and Brazil around 100 years ago, and was first documented in a 3-day-old infant from the New York area in 1934. The severe pathogenic capabilities of Toxoplasma gondii were truly recognized in the 1920s and 1930s, namely in congenitally infected children experiencing a few key symptoms such as hydrocephalus, retinochoroiditis and encephalitis; it was also linked to severe ocular inflammation. It was not until the 1980s, however, when Toxoplasma gondii emerged as a major cause of death in patients—this further illustrated the public health concern in finding a control for the infection. Additionally, researchers first began questioning the potential pathways of the Toxoplasma gondii parasite when it started being reported as an essential factor in New Zealand sheep abortions in the 1950s. Later in the 1960s, an even more essential discovery was made that furthered the research of Toxoplasmosis: cats were the definitive hosts. This finding aided in scientists’ understanding of Toxoplasma gondii’s life cycle, and how the “oocyst stage of [the parasite that was] shed in the faeces of infected cats was found to be an important source of” the disease; the stage is apparently very vigorous and can potentially survive in an environment for up to 12-18 months. This also gave an explanation of why the infection was found in herbivorous animals and individuals who are vegetarians. Toxoplasmosis is a global disease that can essentially infect all warm-blooded hosts, and due to its high prevalence rate in certain areas of the world, it continues to pose a significant health concern.
Figure 1. Mechanism of Isoleucine-tRNA synthetase, putative in enzyme assay.
Enzyme Assay information: “Reactions were performed in a 50μl volume with rIleRS enzyme (40 μg/ml), L-isoleucine (0.1, 0.5, 1, 5, 10 and 50 mM), 8 mu;M of tRNAIle, 2u/ml PPiase, 100μM ATP, and 1mM DTT in aminoacylation buffer. Reactions were incubated at 37°C; at 2.5, 5 and 10 minutes, 15 μl aliquots were withdrawn and mixed with 10 mM EDTA on ice to stop the reactions. 100 μl of malachite green solution was added, and the absorbance measured at 620 nm.”
Link to Sigma (or other company) page for assay: Told not to do
Links to assay reagents (substrates): ATP + L-isoleucine + tRNAIle = AMP + diphosphate + L-isoleucyl-tRNAIle
List cost and quantity of substrate reagents, supplier, and catalog #: vial of ~1 mg ATP (Sigma), FLAAS-5VL: $120.00 L-isoleucine reagent grade, >98% (HPLC) (Sigma-Aldrich), I2752-100G: $127.00 lyophilized powder (Sigma), R6018-1UN: $42.00
Current Inhibitors: “Mupirocin, currently in clinical use for the topical treatment of Staphylococcus aureus, that acts through the inhibition of the isoleucyl-tRNA synthetase” http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940080/
Expression Information (has it been expressed in bacterial cells): Expressed in E. coli cells
Purification Method: Recombinant His-tagged wild-type and mutants enzymes from Escherichia coli by nickel affinity chromatography
Structure (PDB or Homology model): 1ILE Figure 2. PDB protein structure of Isoleucine-tRNA synthetase, putative.
Target (protein/gene name): Isoleucine-tRNA synthetase, putative
NCBI Gene # or RefSeq#: 7896257
Protein ID (NP or XP #) or Wolbachia#: XP_002369568.1
Organism (including strain): Toxoplasma gondii (strain: ME49)
Etiologic Risk Group (see link below):Appendix B-III-A. Risk Group 3 (RG3) - Bacterial Agents
Disease Information:
Toxoplasmosis is a resourceful and global infection that results from the intracellular protozoan parasite Toxoplasma gondii. The disease was discovered by scientists researching in North Africa and Brazil around 100 years ago, and was first documented in a 3-day-old infant from the New York area in 1934. The severe pathogenic capabilities of Toxoplasma gondii were truly recognized in the 1920s and 1930s, namely in congenitally infected children experiencing a few key symptoms such as hydrocephalus, retinochoroiditis and encephalitis; it was also linked to severe ocular inflammation. It was not until the 1980s, however, when Toxoplasma gondii emerged as a major cause of death in patients—this further illustrated the public health concern in finding a control for the infection.
Additionally, researchers first began questioning the potential pathways of the Toxoplasma gondii parasite when it started being reported as an essential factor in New Zealand sheep abortions in the 1950s. Later in the 1960s, an even more essential discovery was made that furthered the research of Toxoplasmosis: cats were the definitive hosts. This finding aided in scientists’ understanding of Toxoplasma gondii’s life cycle, and how the “oocyst stage of [the parasite that was] shed in the faeces of infected cats was found to be an important source of” the disease; the stage is apparently very vigorous and can potentially survive in an environment for up to 12-18 months. This also gave an explanation of why the infection was found in herbivorous animals and individuals who are vegetarians. Toxoplasmosis is a global disease that can essentially infect all warm-blooded hosts, and due to its high prevalence rate in certain areas of the world, it continues to pose a significant health concern.
Link to TDR Targets page: http://www.tdrtargets.org/targets/view?gene_id=263560
Link to Gene Database page: http://www.ncbi.nlm.nih.gov/gene/7896257
Essentiality of this protein: Very essential in the biosynthesis and metabolism of proteins
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940080/pdf/main.pdf http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4000095/pdf/zek504.pdf
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer (1 amino acid chain and 2 ligands in ASU)
Complex of proteins?: Monomer in solutions
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism):
http://www.ncbi.nlm.nih.gov/pubmed/11327600
http://www.sciencedirect.com/science/article/pii/S0960894X05006049
http://www.ncbi.nlm.nih.gov/pubmed/9554847?dopt=Abstract ?
EC#: 6.1.1.5
Link to BRENDA EC# page: http://www.brenda-enzymes.org/enzyme.php?ecno=6.1.1.5
Figure 1. Mechanism of Isoleucine-tRNA synthetase, putative in enzyme assay.
Enzyme Assay information: “Reactions were performed in a 50μl volume with rIleRS enzyme (40 μg/ml), L-isoleucine (0.1, 0.5, 1, 5, 10 and 50 mM), 8 mu;M of tRNAIle, 2u/ml PPiase, 100μM ATP, and 1mM DTT in aminoacylation buffer. Reactions were incubated at 37°C; at 2.5, 5 and 10 minutes, 15 μl aliquots were withdrawn and mixed with 10 mM EDTA on ice to stop the reactions. 100 μl of malachite green solution was added, and the absorbance measured at 620 nm.”
Link to Sigma (or other company) page for assay: Told not to do
Link (or citation) to paper that contains assay information:
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3774597/
Links to assay reagents (substrates):
ATP + L-isoleucine + tRNAIle = AMP + diphosphate + L-isoleucyl-tRNAIle
List cost and quantity of substrate reagents, supplier, and catalog #:
vial of ~1 mg ATP (Sigma), FLAAS-5VL: $120.00
L-isoleucine reagent grade, >98% (HPLC) (Sigma-Aldrich), I2752-100G: $127.00
lyophilized powder (Sigma), R6018-1UN: $42.00
Current Inhibitors: “Mupirocin, currently in clinical use for the topical treatment of Staphylococcus aureus, that acts through the inhibition of the isoleucyl-tRNA synthetase”
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3940080/
Expression Information (has it been expressed in bacterial cells): Expressed in E. coli cells
Purification Method: Recombinant His-tagged wild-type and mutants enzymes from Escherichia coli by nickel affinity chromatography
Structure (PDB or Homology model): 1ILE
Figure 2. PDB protein structure of Isoleucine-tRNA synthetase, putative.
Amino Acid Sequence: MFKEVGEPNFPKLEEEVLAFWKREKIFQKSVENRKGGPRYTVYEGPPTANGLPHVGHAQARSYKDLFPRYKTMRGYYAPR
RAGWDTHGLPVELEVEKKLGLKSKREIEAYGIERFNQACRESVFTYEKEWEAFTERIAYWVDLEDAYATLEPTYIESIWW
SLKNLFDRGLLYRDHKVVPYCPRCGTPLSSHEVALGYKEIQDPSVYVRFPLKEPKKLGLEKASLLIWTTTPWTLPGNVAA
AVHPEYTYAAFQVGDEALILEEGLGRKLLGEGTQVLKTFPGKALEGLPYTPPYPQALEKGYFVVLADYVSQEDGTGIVHQ
APAFGAEDLETARVYGLPLLKTVDEEGKLLVEPFKGLYFREANRAILRDLRGRGLLFKEESYLHSYPHCWRCSTPLMYYA
TESWFIKNTLFKDELIRNNQEIHWVPPHIKEGRYGEWLKNLVDWALSRNRYWGTPLPIWVCQACGKEEAIGSFQELKARA
TKPLPEPFDPHRPYVDQVELACACGGTMRRVPYVIDVWYDSGAMPFASLHYPFEHEEVFRESFPADFIAEGIDQTRGWFN
SLHQLGVMLFGSIAFKNVICHGLILDEKGQKMSKSKGNVVDPWDIIRKFGADALRWYIYVSAPPEADRRFGPNLVRETVR
DYFLTLWNVYSFFVTYANLDRPDLKNPPPPEKRPEMDRWLLARMQDLIQRVTEALEAYDPTTSARALRDFVVEDLSQWYV
RRNRRRFWKNEDALDREAAYATLYEALVLVATLAAPFTPFLAEVLWQNLVRSVRLEAKESVHLADWPEADPALADEALVA
QMRAVLKVVDLARAARAKSGV
Molecular Weight: 94813.56 kD
Length: 821
Extinction coefficients:
Extinction coefficients are in units of M-1 cm-1, at 280 nm measured in water.
Ext. coefficient 200705
Abs 0.1% (=1 g/l) 2.123, assuming all pairs of Cys residues form cystines
Ext. coefficient 200080
Abs 0.1% (=1 g/l) 2.116, assuming all Cys residues are reduced
TMpred graph Image:
Figure 3. TMpred graph of Isoleucine-tRNA synthetase, putative.
CDS Gene Sequence:
atgtttaaagaagtgggcgaaccgaactttccgaaactggaagaagaagtgctggcgttt
tggaaacgcgaaaaaatttttcagaaaagcgtggaaaaccgcaaaggcggcccgcgctat
accgtgtatgaaggcccgccgaccgcgaacggcctgccgcatgtgggccatgcgcaggcg
cgcagctataaagatctgtttccgcgctataaaaccatgcgcggctattatgcgccgcgc
cgcgcgggctgggatacccatggcctgccggtggaactggaagtggaaaaaaaactgggc
ctgaaaagcaaacgcgaaattgaagcgtatggcattgaacgctttaaccaggcgtgccgc
gaaagcgtgtttacctatgaaaaagaatgggaagcgtttaccgaacgcattgcgtattgg
gtggatctggaagatgcgtatgcgaccctggaaccgacctatattgaaagcatttggtgg
agcctgaaaaacctgtttgatcgcggcctgctgtatcgcgatcataaagtggtgccgtat
tgcccgcgctgcggcaccccgctgagcagccatgaagtggcgctgggctataaagaaatt
caggatccgagcgtgtatgtgcgctttccgctgaaagaaccgaaaaaactgggcctggaa
aaagcgagcctgctgatttggaccaccaccccgtggaccctgccgggcaacgtggcggcg
gcggtgcatccggaatatacctatgcggcgtttcaggtgggcgatgaagcgctgattctg
gaagaaggcctgggccgcaaactgctgggcgaaggcacccaggtgctgaaaacctttccg
ggcaaagcgctggaaggcctgccgtataccccgccgtatccgcaggcgctggaaaaaggc
tattttgtggtgctggcggattatgtgagccaggaagatggcaccggcattgtgcatcag
gcgccggcgtttggcgcggaagatctggaaaccgcgcgcgtgtatggcctgccgctgctg
aaaaccgtggatgaagaaggcaaactgctggtggaaccgtttaaaggcctgtattttcgc
gaagcgaaccgcgcgattctgcgcgatctgcgcggccgcggcctgctgtttaaagaagaa
agctatctgcatagctatccgcattgctggcgctgcagcaccccgctgatgtattatgcg
accgaaagctggtttattaaaaacaccctgtttaaagatgaactgattcgcaacaaccag
gaaattcattgggtgccgccgcatattaaagaaggccgctatggcgaatggctgaaaaac
ctggtggattgggcgctgagccgcaaccgctattggggcaccccgctgccgatttgggtg
tgccaggcgtgcggcaaagaagaagcgattggcagctttcaggaactgaaagcgcgcgcg
accaaaccgctgccggaaccgtttgatccgcatcgcccgtatgtggatcaggtggaactg
gcgtgcgcgtgcggcggcaccatgcgccgcgtgccgtatgtgattgatgtgtggtatgat
agcggcgcgatgccgtttgcgagcctgcattatccgtttgaacatgaagaagtgtttcgc
gaaagctttccggcggattttattgcggaaggcattgatcagacccgcggctggtttaac
agcctgcatcagctgggcgtgatgctgtttggcagcattgcgtttaaaaacgtgatttgc
catggcctgattctggatgaaaaaggccagaaaatgagcaaaagcaaaggcaacgtggtg
gatccgtgggatattattcgcaaatttggcgcggatgcgctgcgctggtatatttatgtg
agcgcgccgccggaagcggatcgccgctttggcccgaacctggtgcgcgaaaccgtgcgc
gattattttctgaccctgtggaacgtgtatagcttttttgtgacctatgcgaacctggat
cgcccggatctgaaaaacccgccgccgccggaaaaacgcccggaaatggatcgctggctg
ctggcgcgcatgcaggatctgattcagcgcgtgaccgaagcgctggaagcgtatgatccg
accaccagcgcgcgcgcgctgcgcgattttgtggtggaagatctgagccagtggtatgtg
cgccgcaaccgccgccgcttttggaaaaacgaagatgcgctggatcgcgaagcggcgtat
gcgaccctgtatgaagcgctggtgctggtggcgaccctggcggcgccgtttaccccgttt
ctggcggaagtgctgtggcagaacctggtgcgcagcgtgcgcctggaagcgaaagaaagc
gtgcatctggcggattggccggaagcggatccggcgctggcggatgaagcgctggtggcg
cagatgcgcgcggtgctgaaagtggtggatctggcgcgcgcggcgcgcgcgaaaagcggc
gtg
GC% Content for gene: 57.2473
CDS Gene Sequence (codon optimized): Told not to do until target is selected
GC% Content for gene (codon optimized): Told not to do until target is selected
PDB # or closest PDB entry if using homology model: 10.2210/pdb1ile/pdb (1ILE) http://www.rcsb.org/pdb/explore/explore.do?structureId=1ile
(target from different source organism that is similar in structure according to PDB: http://sbkb.org/tt/search?targetid=RSGI-ttk003000883.1&lab=RSGI ; source organism is Thermus thermophilus)
For Homology Model option: http://www.uniprot.org/uniprot/Q4QG44
Show pairwise alignment of BLASTP search in NCBI against the PDB: http://blast.ncbi.nlm.nih.gov/Blast.cgi
Query 1 MFKEVGEPNFPKLEEEVLAFWKREKIFQKSVENRKGGPRYTVYEGPPTANGLPHVGHAQA 60
MFKEVGEPNFPKLEEEVLAFWKREKIFQKSVENRKGGPRYTVYEGPPTANGLPHVGHAQA
Sbjct 1 MFKEVGEPNFPKLEEEVLAFWKREKIFQKSVENRKGGPRYTVYEGPPTANGLPHVGHAQA 60
Query 61 RSYKDLFPRYKTMRGYYAPRRAGWDTHGLPVELEVEKKLGLKSKREIEAYGIERFNQACR 120
RSYKDLFPRYKTMRGYYAPRRAGWDTHGLPVELEVEKKLGLKSKREIEAYGIERFNQACR
Sbjct 61 RSYKDLFPRYKTMRGYYAPRRAGWDTHGLPVELEVEKKLGLKSKREIEAYGIERFNQACR 120
Query 121 ESVFTYEKEWEAFTERIAYWVDLEDAYATLEPTYIESIWWSLKNLFDRGLLYRDHKVVPY 180
ESVFTYEKEWEAFTERIAYWVDLE+AYATLEPTYIESIWWSLKNLFDRGLLYRDHKVVPY
Sbjct 121 ESVFTYEKEWEAFTERIAYWVDLENAYATLEPTYIESIWWSLKNLFDRGLLYRDHKVVPY 180
Query 181 CPRCGTPLSSHEVALGYKEIQDPSVYVRFPLKEPKKLGLEKASLLIWTTTPWTLPGNVAA 240
CPRCGTPLSSHEVALGYKEIQDPSVYVRFPLKEPKKLGLEKASLLIWTTTPWTLPGNVAA
Sbjct 181 CPRCGTPLSSHEVALGYKEIQDPSVYVRFPLKEPKKLGLEKASLLIWTTTPWTLPGNVAA 240
Query 241 AVHPEYTYAAFQVGDEALILEEGLGRKLLGEGTQVLKTFPGKALEGLPYTPPYPQALEKG 300
AVHPEYTYAAFQVGDEALILEEGLGRKLLGEGT VLKTFPGKALEGLPYTPPYPQALEKG
Sbjct 241 AVHPEYTYAAFQVGDEALILEEGLGRKLLGEGTPVLKTFPGKALEGLPYTPPYPQALEKG 300
Query 301 YFVVLADYVSQEDGTGIVHQAPAFGAEDLETARVYGLPLLKTVDEEGKLLVEPFKGLYFR 360
YFVVLADYVSQEDGTGIVHQAPAFGAEDLETARVYGLPLLKTVDEEGKLLVEPFKGLYFR
Sbjct 301 YFVVLADYVSQEDGTGIVHQAPAFGAEDLETARVYGLPLLKTVDEEGKLLVEPFKGLYFR 360
Query 361 EANRAILRDLRGRGLLFKEESYLHSYPHCWRCSTPLMYYATESWFIKNTLFKDELIRNNQ 420
EANRAILRDLRGRGLLFKEESYLHSYPHCWRCSTPLMYYATESWFIKNTLFKDELIR NQ
Sbjct 361 EANRAILRDLRGRGLLFKEESYLHSYPHCWRCSTPLMYYATESWFIKNTLFKDELIRKNQ 420
Query 421 EIHWVPPHIKEGRYGEWLKNLVDWALSRNRYWGTPLPIWVCQACGKEEAIGSFQELKARA 480
EIHWVPPHIKEGRYGEWLKNLVDWALSRNRYWGTPLPIWVCQACGKEEAIGSFQELKARA
Sbjct 421 EIHWVPPHIKEGRYGEWLKNLVDWALSRNRYWGTPLPIWVCQACGKEEAIGSFQELKARA 480
Query 481 TKPLPEPFDPHRPYVDQVELACACGGTMRRVPYVIDVWYDSGAMPFASLHYPFEHEEVFR 540
TKPLPEPFDPHRPYVDQVELACACGGTMRRVPYVIDVWYDSGAMPFASLHYPFEHEEVFR
Sbjct 481 TKPLPEPFDPHRPYVDQVELACACGGTMRRVPYVIDVWYDSGAMPFASLHYPFEHEEVFR 540
Query 541 ESFPADFIAEGIDQTRGWFNSLHQLGVMLFGSIAFKNVICHGLILDEKGQKMSKSKGNVV 600
ESFPADFIAEGIDQTRGWFN LHQLGVMLFGSIAFKNVICHGLIL E GQKMSKSKGNVV
Sbjct 541 ESFPADFIAEGIDQTRGWFNFLHQLGVMLFGSIAFKNVICHGLILYEMGQKMSKSKGNVV 600
Query 601 DPWDIIRKFGADALRWYIYVSAPPEADRRFGPNLVRETVRDYFLTLWNVYSFFVTYANLD 660
DPWDIIR+FGADALRWYIYVSAPPEADRRFGPNLVRETVRDYFLTLWNVYSFFVTYANLD
Sbjct 601 DPWDIIREFGADALRWYIYVSAPPEADRRFGPNLVRETVRDYFLTLWNVYSFFVTYANLD 660
Query 661 RPDLKNPPPPEKRPEMDRWLLARMQDLIQRVTEALEAYDPTTSARALRDFVVEDLSQWYV 720
RPDLKNPPPPEKRPEMDRWLLARMQDLIQRVTEALEAYDPTTSARALRDFVVEDLSQWYV
Sbjct 661 RPDLKNPPPPEKRPEMDRWLLARMQDLIQRVTEALEAYDPTTSARALRDFVVEDLSQWYV 720
Query 721 RRNRRRFWKNEDALDREAAYATLYEALVLVATLAAPFTPFLAEVLWQNLVRSVRLEAKES 780
RRNRRRFWKNEDALDREAAYATLYEALVLVATLAAPFTPFLAEVLWQNLVRSVR EAKES
Sbjct 721 RRNRRRFWKNEDALDREAAYATLYEALVLVATLAAPFTPFLAEVLWQNLVRSVRPEAKES 780
Query 781 VHLADWPEADPALADEALVAQMRAVLKVVDLARAARAKSGV 821
VHLADWPEADPALADEALVAQMRAVLKVVDLARAARAKSGV
Sbjct 781 VHLADWPEADPALADEALVAQMRAVLKVVDLARAARAKSGV 821
Query Coverage: 100%
Max % Identities: (813/821) 99%
% Positives: (815/821) 99%
Chain used for homology:
MFKEVGEPNFPKLEEEVLAFWKREKIFQKSVENRKGGPRYTVYEGPPTANGLPHVGHAQARSYKDLFPRYKTMRGYYAPRRAGWDTHGLPVELEVEKKLGLKSKREIEAYGIERFNQACRESVFTYEKEWEAFTERIAYWV
DLENAYATLEPTYIESIWWSLKNLFDRGLLYRDHKVVPYCPRCGTPLSSHEVALGYKEIQDPSVYVRFPLKEPKKLGLEKASLLIWTTTPWTLPGNVAAAVHPEYTYAAFQVGDEALILEEGLGRKLLGEGTPVLKTFPGK
ALEGLPYTPPYPQALEKGYFVVLADYVSQEDGTGIVHQAPAFGAEDLETARVYGLPLLKTVDEEGKLLVEPFKGLYFREANRAILRDLRGRGLLFKEESYLHSYPHCWRCSTPLMYYATESWFIKNTLFKDELIRKNQEIH
WVPPHIKEGRYGEWLKNLVDWALSRNRYWGTPLPIWVCQACGKEEAIGSFQELKARATKPLPEPFDPHRPYVDQVELACACGGTMRRVPYVIDVWYDSGAMPFASLHYPFEHEEVFRESFPADFIAEGIDQTRGWFNFLHQ
LGVMLFGSIAFKNVICHGLILYEMGQKMSKSKGNVVDPWDIIREFGADALRWYIYVSAPPEADRRFGPNLVRETVRDYFLTLWNVYSFFVTYANLDRPDLKNPPPPEKRPEMDRWLLARMQDLIQRVTEALEAYDPTTSAR
ALRDFVVEDLSQWYVRRNRRRFWKNEDALDREAAYATLYEALVLVATLAAPFTPFLAEVLWQNLVRSVRPEAKESVHLADWPEADPALADEALVAQMRAVLKVVDLARAARAKSGVKTRTPLPLLLVTAPTALEREGLKRF
AHEIAEELNVKEVRVLEPGEEILSYRVLPNLKLLGRKYGKLVPKIREALQRERERAAALALKGEAIPLEVEGEALTLLPEEVLLEAEAPKGYQALEKDGYVAALKVEVTEALRMEGLARDLIRLLQQARKDMGLKVSDRIR
VGYEAEGPYLEALKRHGPWIAEEVLATAFGEGLFGGFEARVEDEEGKAVFHLARAE
Primer design results for 'tail' primers (this is just 2 sequences):Told not to do until target is selected